1. Field of the Invention
The present invention generally relates to the field of medical screening and diagnostic hematology instruments which performs complete blood count (CBC), white blood cell 3 part differential and other blood parameter measurements for humans, or similar analysis for other cells, or similar analysis for animal blood, or similar analysis of material components that have requirements for its particle contents and size—such as food, beverage, cosmetics, pharmaceutical or petroleum with a need to test the purity of said substance. More particularly, the invention relates to the methods and instruments for differentiating and identifying subpopulations of leukocytes in a blood sample by using Coulter impedance and light scattering only, especially by using backscattering signals through the new innovative invention of direct coaxial illumination of the Coulter aperture.
2. Background of the Invention
Human blood consists of plasma (approximate 58%) and cellular elements (approximate 42%). The cellular elements include red blood cells (RBC, 4-5 millions per microliter), white blood cells (WBC, 5-9 thousands per microliter), and platelets (PLT, 200-400 thousands per microliter). The WBC primarily have 5 subpopulations: Neutrophils (phagocytosis and proteolysis), Basophils (Inflammation and allergic reactions), Eosinophils (Viral infection), Monocytes (Phagocytosis) Lymphocytes (Including B cells and T cells, function in humoral immunity).
The counting and sizing of RBCs, the counting of WBCs, and counting of platelets is referred to as a complete blood count (CBC). The separation of white blood cells into the five major subpopulations and their quantification on a percentage basis is referred to as a five-part differential (5-diff).
Before 1950 blood cell counts by manual counting used the hemocytometer. The era of automated cell counting was introduced by Wallace H. Coulter in the early 1950's. The Coulter Principle, the electronic method of counting and measuring the size of microscopic particles and named for its inventor Wallace H. Coulter, states that a particle pulled through an orifice (aperture), concurrent with an electrical current, will produce a change in impedance that is proportional to the size of the particle traversing the orifice. This impedance change creates an electric pulse and is commonly referred to as a DC (direct current) signal. The related U.S. Pat. No. 2,656,508 to Coulter was issued Oct. 20, 1953. Although more than half century has passed the Coulter principle and Coulter aperture continue to be used in all hematology blood analyzer today and the DC signal still is the indispensible signal among others. Today ninety-eight percent of CBCs are performed on instruments using the Coulter Principle.
Automated counting increased the sample size of the blood test 100 times more than the usual manual microscope method by counting in excess of 6000 cells per second. Additionally, it decreased the time it took to analyze from 30 minutes to fifteen seconds and reduced the error by a factor of approximately 10 times.
The Coulter Principle states that particle suspension in a weak electrolyte solution of two chambers, separating two electrodes, are connected by a small aperture. When the particles suspended in solution are drawn through the aperture, the particle displaces its volume of electrolyte and momentarily increasing the impedance of the aperture. The Coulter Principle used in impedance-based blood analyzer is schematically described in FIG. 1. The two Chambers, aperture bath 1 and aperture tube 2, are connected by only one path, the aperture 5. Suspended in saline solution 3, blood cell 4, one at a time, due to pressure difference of vacuum 9, passes through the aperture 5 and the impedance changes creates a pulse current 8 between two electrodes 6 and 7. The pulse (DC) is directly proportional to the volume of the cell that produces this pulse. Because Neutrophil, Basophil and Eosinophil have similar volume, by the impedance method, only three parts can be distinguished. Thus, three-part differential (3-diff) blood count can be obtained.
In order to obtain 5-diff, several techniques had emerged since the 90's. Light scattering, radio frequency and chemical dyeing were applied to hematology instruments to obtain five-part differential, and 5-diff hematology instruments available commercially since then. All these instruments are based the technology called flow cytometry.
The basic principle of the flow cytometry based on light scattering blood analyzer can be illustrated as FIG. 2. The two cones of flow cell 10 for the sheath flow 14, force sample flow 15 at the center of sheath flow to form hydrodynamic focusing. Two electrodes 12 and aperture current 13 create an impedance pulse when a cell passes through aperture 11. The laser source 16, through focusing lens 17 illuminates the aperture and creates the forward scattering (FLS) and axial light loss (ALL) detected by detector 19, and 90° side light scattering (SLS) (and fluorescence) detected by detector 18. Thus, when each cell passing the orifice, at least 3 signals can be detected, i.e. DC, FLS and SLS, if a radio frequency source is applied to the two electrodes 12, then another signal, called RF (radio frequency), will be available and RF is sensitive to cell internal contents.
RF is Wallace Coulter's another important invention and related U.S. Pat. No. 3,502,974 to Coulter was issued Mar. 24, 1970. FIG. 3 is a schematic diagram of flow cell. In the center of quartz rectangular prism 25, size about 4 mm×4 mm×8 mm, drill a through hole of diameter 50 μm, along its long axis 30. Two cones 22 were made by a special drilling grinding machine from opposite top surface 28 and bottom surface 29, aligned cone axis and roof to coincide with the 50 μm hole axis 30 until 50 μm orifice 23 about 70 μm long in the middle of the rectangular prism. Two cones 22 for the sheath flow 21 force the sample flow 24 in the center of orifice 23 to form a sample filament so that cell will passing through the orifice one at a time (hydrodynamic focusing). The focused laser beam 20 illuminates the cell in the sample flow. From FIG. 2 and FIG. 3 we can see that in the flow cytometry based instruments, the laser light beam propagates along the axis in the direction (horizontal in the FIGs) perpendicular to cell moving direction (vertical in the FIGs), which will be referred to as cross illumination in this paper to distinguish from the coaxial illumination presented in this invention, which will be described later.
All flow cytometry based light scattering hematology instruments in the world now are all using flow cell, hydrodynamic focusing and cross illumination. When a cell passes through an orifice and interacts with focused laser light, the light scattering signals are created and can be detected. Except forward light scattering (FLS), axial light loss (ALL), and 90° side light scattering (SLS) signals shown in FIG. 2, there will have back scattering light (BSL) which also can be detected. Although back scattering is proved more sensitive to particle's (cell's) internal content and structure (Kerker et al., 1979; Kerker, 1983; Mourant et al., 1998), its intensity is about 3 orders of magnitude weaker than forward scattering. When using flow cell as shown in FIG. 2 and FIG. 3, the focused laser beam will first hit flow cell's front surface 26, then hit the cylindrical surface of orifice 23, and finally hit the back surface 27, thus very strong reflected light creates unwanted background for back scattering detection. As Gangstead et al., pointed out in U.S. Pat. No. 6,646,742, “Unfortunately, using any of the available conventional flow cytometry illumination arrangements, the walls of these walled-conduit structures conventionally generates such an enormous quantity of background scatter noise”. This is the one of the main technical bottleneck that no commercial hematology instrument is capable of incorporating backscattering for enhancing cell discrimination power purpose.
Several attempts were made using backscattering only in the laboratory, such as U.S. Pat. No. 6,743,634 B2 (Kramer, Jun. 1, 2004) and U.S. Pat. No. 6,869,569 B2 (Kramer, Mar. 22, 2005), all use a plurality of optical fibers and PMTs and are impossible of being incorporated into a practical commercial instrument due to its complex structure and cost.
Only this unique invention will make detection of cell's backscattering signal possible in a practical commercial hematology instrument for the first time in the hematology instrument history.